The present invention relates to a concentration device using magnetic particles and a method therefor. More particularly, the present invention relates to a concentration device using magnetic particles and a concentration method using magnetic particles applied to medical treatment, examination, diagnosis, treatment, research, quantitative, qualitative, measurement or the like, in which concentration is automatically and reliably increased so that a target substance such as a microscopic amount of bacteria to be suspended in a large volume of liquid, is concentrated to thereby be suspended in a small volume of liquid, in order to perform processing such as separation, extraction or recovery of, for example, immunity substances such as antibodies or antigens, genetic substances (DNA, RNA, mRNA, oligonucleotide or the like), biopolymers such as proteins or hormone substances, organisms such as bacteria, viruses, cells, or other useful compounds such as medical drugs, in various fields such as the engineering field, medical field, agricultural field, scientific field and pharmaceutical science field.
Recently, the need for performing various processing such as separation, extraction, inspection or measurement of target substances such as organisms or biopolymers of bacteria or the like (for example, aqueous dissolved bacteria such as 0-157, cryptolipolysium, salmonella or the like, genetic materials such as DNA) is increasing.
For the purpose thereof, it is necessary to perform various processing after having cultured the bacteria, or in the case of genetic materials or the like, after having increased the concentration of the target substance by performing amplification.
The target substance such as bacteria or genetic materials is naturally obtained with a small amount thereof suspended in a large volume (for example, several liters) of liquid. In order to perform culture or amplification for this target substance, it is necessary to extract or separate the target substance from the suspension.
Therefore, separation and extraction has been heretofore performed by passing a large volume of suspension containing the target substance suspended therein through a filter. Normally, however, many impurities are contained other than the target substance in the suspension. In the case of a membrane filter or the like, these are likely to cause clogging due to the impurity and fouling. Hence this has the problem in that it is difficult or it requires time and labor to separate the target substance from a large volume of suspension.
Moreover, considering a case where a substance such as bacteria that affect organisms such as humans is handled, there is a demand for an apparatus which can automatically perform processing without requiring human intervention, which makes it possible to replace a part coming in contact with the suspension, and which does not require cleaning and can be used efficiently and reliably.
It is therefore a first object of the present invention to provide a concentration device using magnetic particles and a concentration method using magnetic particles, which can perform concentration of a large volume of liquid efficiently, quickly and reliably.
It is a second object of the present invention to provide a concentration device using magnetic particles and a concentration method using magnetic particles, wherein concentration, separation or extraction processing is not affected by the existence of impurities.
It is a third object of the present invention to provide a concentration device using magnetic particles and a concentration method using magnetic particles, wherein the part coming in contact with the suspension can have a construction such that a container or a disposable tip can be replaced, and a simple mechanism is used to thereby make it low-priced and easy to handle, without requiring cleaning.
It is a fourth object of the present invention to provide a concentration device using magnetic particles and a concentration method using magnetic particles, which are suitable for automated processing and consistent processing without requiring human intervention.
It is a fifth object of the present invention to provide a concentration device using magnetic particles and a concentration method using magnetic particles, suitable for handling microorganisms such as bacteria and biopolymers such as DNA substances. More particularly, it is an object of the present invention to provide a concentration device using magnetic particles and a concentration method using magnetic particles, suitable for amplification by a PCR method for genetic materials such as DNA.
In order to solve the above technical problems, a first aspect of the present invention is characterized by comprising: a liquid suction passage in which liquid can pass through only in a suction direction; a liquid discharge passage in which liquid can pass through only in a discharge direction; a magnetic force device which can exert a magnetic field from outside of the liquid passage on at least one liquid passage thereof or remove the magnetic field, and which can separate magnetic particles having directly or indirectly captured a target substance suspended in the liquid by having the magnetic particles adhere to the inner wall of the liquid passage; a storage section communicated with each liquid passage, for storing the sucked liquid; and a pressure adjustment device for sucking and discharging the liquid by adjusting the pressure in the storage section.
The term xe2x80x9cdirectly capturing the target substancexe2x80x9d herein stands for capturing the target substance by bonding the target substance such as bacteria with the magnetic particles. In order to do that, it is necessary that the magnetic particles have a substance for bonding with the target substance by means of a coating or the like. For example, there can be mentioned a case where glass is coated on the magnetic particles, and genetic materials such as DNA are adsorbed on the glass and captured.
The term xe2x80x9cindirectly capturing the target substancexe2x80x9d herein stands for a case where magnetic particles, target substance and a carrier originally existing separately and independently are suspended in liquid, and the carrier bonds with the magnetic particles and the target substance, so that magnetic particles capture the target substance. As the carrier, for example, there are gel substances such as hydroxyapatite and a cellulose carrier. When a gel substance such as hydroxyapatite is used as a carrier, the gel substance takes both the magnetic particles and the target substance into the inside thereof to thereby capture the target substance. When the cellulose carrier is used as a carrier, magnetic particles and the target substance are adhered to holes and gaps in the cellulose carrier to thereby capture the target substance. Capture can be effected by putting the magnetic particles or magnetic particles and the carrier into the liquid containing the target substance, and mixing or stirring these. The target substance captured by the magnetic particles can be dissolved by physical processing such as stirring or heating, or can be eluted or alienated from the magnetic particles by chemical processing such as by adjusting the pH to dissolve the target substance as with the hydroxyapatite, or by mixing with a reagent. The liquid suction passage and the liquid discharge passage are capable of passing the liquid only in the suction direction or in the discharge direction of the liquid by, for example, a check valve. The check valve may be provided in a part of the liquid suction passage and the liquid discharge passage, or may be provided outside the liquid suction passage and the liquid discharge passage, for example, attached to the storage section. When the suction port and the discharge port of the liquid passage are inserted into different containers, the suspension before separating the magnetic particles and the residual liquid after the separation thereof can be separated, thereby enabling efficiency improvement of the processing. When these ports are inserted into the same container, the liquid passage forms a circuit, thereby enabling separation to be repeated without replacing the suction port and the discharge port.
According to the first aspect, at the time of repeating a simple operation such as passing a large volume of suspension through the liquid passage, or sucking the liquid from the liquid suction passage and discharging the liquid from the liquid discharge passage, with respect to all of the suspension, the magnetic particles in the suspension are automatically separated. Hence, all of the magnetic particles in the suspension can be efficiently and easily separated with a simple construction and without leakage.
According to the present invention, by re-suspending the separated magnetic particles in an optional amount of liquid, concentration to an optional proportion can be performed. Hence, the culture process and the amplification process thereafter can be made efficient, and the density of a large volume of suspension in which the target substance is suspended in a dilute amount can be increased efficiently or within a short period of time.
According to the present invention, the target substance can be captured by magnetic particles, and the liquid can be made to pass through the liquid passage, with the magnetic particles attracted to the liquid passage and separated, by exerting a magnetic field from outside of the liquid passage. Hence, since impurities can be eliminated by the suction and discharge operation, separation, extraction or the like of the target substance is not hindered.
Also, in the case where the magnetic particles capture the target substance indirectly via a carrier, it is not necessary to perform processing such as coating with respect to the magnetic particles. Hence processing can be performed at a low cost.
A second aspect of the present invention is characterized in that in the first aspect, the liquid suction passage and the liquid discharge passage are provided in parallel to each other with a space, so as to protrude downward below the storage section, with the pressure adjustment device provided above the storage section. According to the present invention, since the liquid suction passage and the liquid discharge passage are provided in parallel to each other with a space, if a container that can be divided by that space is prepared and each liquid passage is inserted therein, the sucked liquid can be discharged to another container without requiring a shift. According to the second aspect, since the liquid suction passage and the liquid discharge passage are provided so as to protrude downward below the storage section, and the pressure adjustment device is provided above the storage section, the apparatus can be made compact, and concentration processing can be performed efficiently by using a special container.
A third aspect of the present invention is characterized in that in the first aspect or in the second aspect, the liquid suction passage and the liquid discharge passage are formed substantially in a two-stage form with a tip portion having a small diameter and a large-diameter portion, respectively. Hence the liquid passage can be inserted also with respect to a container having a thin opening, and by performing separation of the magnetic particles by the large-diameter portion, clogging or the like can be prevented. According to the third aspect, insertion is also possible with respect to a container having a thin opening, and clogging or the like can be prevented at the time of performing separation of magnetic particles.
A fourth aspect of the present invention is characterized in that in any one of the first aspect to the third aspect, the magnetic force device is obtained by providing a permanent magnet, an electromagnet or a magnetic substance outside of at least one of the liquid passages. In the case of the permanent magnet, by providing this so as to be able to be brought near or alienated from the liquid passage, the magnetic field can be exerted or removed.
Moreover, in the case of the electromagnet, by flowing or cutting off the electric current, the magnetic field is generated or removed. In the case of the magnetic substance, by magnetizing or demagnetizing the magnetic substance, the magnetic field is generated or removed. As a result, the magnetic field can be reliably exerted or removed. According to the fourth aspect, the magnetic field can be reliably exerted on or removed from the inside of the liquid passage.
A fifth aspect of the present invention is characterized in that in any one of the first aspect to the fourth aspect, the storage section has a cylinder, and the pressure adjustment device has a plunger inserted into the cylinder so as to slide therein. Here, in the storage section, it is desirable to control so that the surface of the sucked liquid and the bottom surface of the piston do not come in contact with each other via an air layer. As a result, contact between the mechanical part and the suspension can be avoided, thereby enabling reuse without requiring cleaning. Moreover, it is desirable to provide an O-ring groove around the circumference of the plunger, so as to attach an O-ring formed of a resilient body in that groove. As a result, liquid leakage can be reliably prevented. With the fifth aspect, by controlling so that the surface of the sucked liquid and the bottom surface of the piston do not come in contact with each other via the air layer, in the storage section, reliable processing can be performed without the possibility of contamination.
A sixth aspect of the present invention is characterized in that in any one of the first aspect to the fifth aspect, the pressure adjustment device has an air flow path provided in the storage section, and a pump for performing suction and discharge of a gas in the storage section via the air flow path. As a result, the structure thereof can be made simple and cleaning becomes easy, without requiring a complicated mechanical portion in the storage section. With the sixth aspect, the pressure adjustment device is communicated with the pump performing suction and discharge of a gas in the storage section, via the storage section and the air flow path. Hence, it is not required to provide a complicated mechanical portion in the storage section, thereby simplifying the structure and making cleaning easy.
A seventh aspect of the present invention is characterized in that in any one of the first aspect to the sixth aspect, the storage section is provided detachably with respect to the pressure adjustment device. Here, the liquid suction passage and the liquid discharge passage may be formed integrally with the storage section, or may be provided detachably with respect to the storage section. According to the present invention, since the storage section including the liquid suction passage and the liquid discharge passage is provided detachably with respect to the pressure adjustment device, by attaching a new storage section, processing can be proceeded without cleaning. Hence, rapid processing is possible. According to the seventh aspect, since the storage section is provided detachably with respect to the pressure adjustment device, by attaching a new storage section, processing can be proceeded without cleaning. As a result, this is very efficient.
An eighth aspect of the present invention is characterized in that in the seventh aspect, when the storage section is detached, the pressure adjustment device can be mounted with one pipette tip, in which the liquid can pass through both in the suction direction and the discharge direction. As a result, efficient processing is possible by using the same pressure adjustment device and replacing only a portion contacting with the suspension. With the eighth aspect, in the case where the storage section is detached from the pressure adjustment device, one pipette tip can be mounted on the pressure adjustment device. As a result, efficient processing is possible by using the same pressure adjustment device and replacing only the portion contacting with the suspension.
A ninth aspect of the present invention is characterized in that in any one of the first aspect to the eighth aspect, hydroxyapatite is sintered and secured to the magnetic particles, and the pH of the solution containing the magnetic particles suspended therein is adjusted to change the hydroxyapatite to a sol form or a gel form, to thereby make the hydroxyapatite capture or alienate the target substance. According to the present invention, by a simple operation such as adjustment of pH, the hydroxyapatite is changed to a gel form or a sol form so that the magnetic particles can capture the target substance, or the target substance can be eluted or alienated.
A tenth aspect of the present invention is characterized by comprising: a liquid passage having a suction port and a discharge port, in which liquid can pass therethrough; a magnetic force device which can exert a magnetic field from outside of the liquid passage to inside thereof, which can separate magnetic particles having directly or indirectly captured a target substance suspended in the liquid, by having the magnetic particles adhere to an inner wall of the liquid passage; and a pump provided in the liquid passage, for transferring the liquid along a transfer direction of the liquid in the liquid passage.
As a result, magnetic particles in the suspension can be separated or re-suspended during passing through the liquid passage without leakage, with a simple structure and a simple control.
As the xe2x80x9cpumpxe2x80x9d, for example, a peristaltic pump may be used, in which a part or the whole of the liquid passage is formed of a resilient body, and liquid is transferred by sequentially making the liquid passage contract from the outside along the transfer direction of the liquid.
At that time, since there is no contact between the suspension and the mechanical portion for transfer or the magnetic force device, the device can be reused by merely cleaning or replacing the liquid passage. Contact between the suspension containing the target substance such as bacteria suspended therein and the mechanical portion can be avoided or can be made minimum. Particularly, the resilient body portion may be cut and formed detachably, for example, by making as a cartridge. In such a case, this is taken out together with the magnetic particles attracted to that portion, to thereby perform processing such as re-suspension.
According to the tenth aspect, at the time of repeating a simple operation such as passing a large volume of suspension through the liquid passage, or sucking the liquid from the liquid suction passage and discharging the liquid from the liquid discharge passage, with respect to the whole suspension, the magnetic particles in the suspension are automatically separated. Hence, all of the magnetic particles in the suspension can be efficiently and easily separated with a simple construction and without leakage.
According to the present invention, by re-suspending the separated magnetic particles in an optional amount of liquid, concentration to an optional proportion can be performed.
Also, in the case where the magnetic particles capture the target substance indirectly via a carrier, it is not necessary to perform processing such as coating with respect to the magnetic particles. Hence processing can be performed at a low cost.
An eleventh aspect of the present invention is characterized in that in the tenth aspect, a switchover mechanism for switching over the liquid passage in order to re-suspend and discharge the separated magnetic particles is provided in the middle of the liquid passage. In order to switchover, for example, a rotor having a through hole is rotatably provided in the middle of the liquid passage, and at the time of separating the magnetic particles from the suspension, the rotation position of the rotor is set so as to communicate with the liquid passage, and at the time of re-suspension, the rotation position is set so that passing in both the suction direction and the discharge direction of the liquid is possible, and so as to communicate with another liquid passage (pipette device) having a magnetic force device capable of exerting a magnetic field therein. According to the eleventh aspect, by using a liquid passage for re-suspension which is different from the liquid passage for separation, separation and re-suspension can be effectively performed.
A twelfth aspect of the present invention is characterized in that in the tenth aspect or the eleventh aspect, a whole of the liquid passage or a part of the liquid passage is provided so as to be able to be taken out, while attracting the separated magnetic particles. As a construction capable of being taken out, for example, this may be taken out by removing a member attaching the whole liquid passage to a frame or the like, or only a part of the liquid passage may be provided detachably from other parts of the liquid passage. The removed part may be directly attached to an apparatus for performing processing and then used. According to the twelfth aspect, by taking out the whole or a part of the liquid passage, the separated magnetic particles can be used. Hence, processing such as concentration can be easily performed.
A thirteenth aspect of the present invention is characterized in that in the tenth aspect to the twelfth aspect, hydroxyapatite is sintered and secured to the magnetic particles, and the pH of the solution containing the magnetic particles suspended therein is adjusted to change the hydroxyapatite to a sol form or a gel form, to thereby make the hydroxyapatite capture or alienate the target substance. According to the present invention, by a simple operation such as adjustment of pH, the hydroxyapatite is changed to a gel form or a sol form so that the magnetic particles can capture the target substance, or the target substance can be eluted or alienated.
A fourteenth aspect of the present invention is characterized by comprising: the concentration device described in any one of the first to the thirteenth aspects; a shift device capable of shifting the concentration device itself and/or a container mounted outside of the concentration device; and a control device for controlling the operation of the liquid drive device, the magnetic force device and/or the shift device. As a result, the concentration processing can be automated collectively.
A fifteenth aspect of the present invention is characterized in that in the fourteenth aspect, the container has two tanks into which the suction port of the liquid passage and the discharge port of the liquid passage are respectively inserted, a thickness of a partition section for separating the two tanks being formed thinner than a distance between the suction port and the discharge port, and a bottom portion of each liquid receiving section has a slope of downward inclination toward the partition section. As a result, a large volume of liquid can be sucked completely and discharged. Hence, concentration can be performed quickly and efficiently.
A sixteenth aspect of the present invention is characterized in that in the fourteenth aspect, the control device controls so as to suck from a container storing a first volume of suspension, in which the magnetic particles having directly or indirectly captured the target substance are suspended, and pass the suspension through the liquid passage, with the magnetic force device exerting a magnetic field from outside to the liquid passage, to thereby attract the magnetic particles to the inner wall of the liquid passage and separate the magnetic particles, and so as to insert the suction port and the discharge port into a container where a second volume of liquid is stored, the second volume being smaller than the first volume, and suck and discharge the second volume of liquid to effect re-suspension to thereby concentrate the suspension containing the target substance such as bacteria suspended therein, without exerting the magnetic field by the magnetic force device.
Here, the above separation is continued until all the suspension to be sucked by the liquid passage has been sucked. When there is no more suspension to be sucked, by sucking the discharged liquid by the liquid passage, separation can be further performed. In this manner, by repeating separation, separation of magnetic particles can be performed more reliably.
A seventeenth aspect of the present invention is characterized by comprising: a capture step for capturing a target substance such as bacteria in a suspension directly or indirectly by magnetic particles; a separation step for separating the magnetic particles by exerting a magnetic field from outside of a liquid passage to the inside of the liquid passage to thereby attract the magnetic particles to an inner wall of the passage, at a time of passing a suspension having a first volume and in which the magnetic particles which have captured the target substance are suspended, through the liquid passage; a re-suspension step for re-suspending the magnetic particles which have captured the target substance in the liquid, by passing a liquid having a second volume smaller than the first volume through the liquid passage in with the magnetic particles which have captured the target substance have been separated, in a state with the magnetic field not exerted on the liquid passage; and an elution step for eluting the target substance from the magnetic particles which have captured the target substance suspended in the liquid, and separating only the magnetic particles to obtain a suspension in which the target substance is concentrated.
Here, the elution step for obtaining the suspension in which only the target substance is concentrated is performed by, for example, physical processing such as suspension and stirring, or by chemical processing involving mixing a predetermined reagent or the like.
An eighteenth aspect of the present invention is characterized in that in the seventeenth aspect, the separation step effects separation by sucking the suspension from a container storing a first volume of suspension, in which the magnetic particles having directly or indirectly captured the target substance are suspended, and passing the suspension through the liquid passage, with the magnetic field exerted from outside of the liquid passage, to thereby attract the magnetic particles to the inner wall of each liquid passage. Moreover, the re-suspension step effects re-suspension by sucking and discharging a second volume of liquid to the container where the second volume of liquid is stored, the second volume being smaller than the first volume, without exerting the magnetic field by the magnetic force device.
A nineteenth aspect of the present invention is characterized in that in either one of the seventeenth aspect or the eighteenth aspect, the separation step effects separation by sucking the suspension from a container storing a first volume of suspension in which the magnetic particles which have captured the target substance are suspended, to a storage section via the liquid suction passage, with the magnetic field exerted from outside to the liquid suction passage and the liquid discharge passage by the magnetic force device, and discharging the suspension from the storage section via the liquid discharge passage, to thereby attract the magnetic particles to the inner wall of each liquid passage. Moreover, the re-suspension step effects re-suspension by inserting the liquid suction passage and the liquid discharge passage into a container where a second volume of liquid is stored, the second volume being smaller than the first volume and substantially the same amount as that of the storage section, to thereby suck and discharge the second volume of liquid, without exerting the magnetic field by the magnetic force device.
The reason why the second volume of liquid is xe2x80x9csmaller than the first volume and substantially the same amount as that of the storage sectionxe2x80x9d is that if a liquid amount is used which is sufficient for filling the storage section, being the maximum amount of liquid which can be added to the separated magnetic particles with one suction or one discharge by the apparatus, in order to efficiently and reliably re-suspend the magnetic particles attracted to the inner wall of each liquid passage of the concentration device and separated, the magnetic particles can be most efficiently stripped off from the inner wall to effect the re-suspension.
A twentieth aspect of the present invention is characterized in that in any one of the seventeenth aspect to the nineteenth aspect, in a container storing the suspension re-suspended in the re-suspension step, the magnetic particles in the suspension are separated and then re-suspended in a liquid having a third volume smaller than the second volume, by sucking and discharging the suspension, with a magnetic field exerted on the liquid passage, by means of a pipette apparatus having a liquid passage in which liquid can pass through both in the suction direction and the discharge direction of the liquid, and a storage section communicated with the passage and having a capacity smaller than the second volume, and also having a magnetic force device for exerting and removing a magnetic field to/from the liquid passage from outside of the liquid passage.
A twenty-first aspect of the present invention is characterized in that in any one of the seventeenth aspect to the twentieth aspect, the separation step shifts all of the liquid stored in a second container to a first container, after having shifted all of the suspension stored in the first container to the second container, by sucking the liquid stored in the second container via the liquid passage, and discharging the liquid to the first container via the liquid passage, with a magnetic field exerted on the liquid passage from outside.
A twenty-second aspect of the present invention is characterized in that in any one of the seventeenth aspect to the twenty-first aspect, the separation step shifts all of the liquid stored in a second container to a first container, after having shifted all of the suspension stored in the first container to the second container, by sucking the liquid stored in the second container via a liquid suction passage, and discharging the liquid to the first container via a liquid discharge passage, with a magnetic field exerted on the liquid suction passage and the liquid discharge passage from outside. As a result, magnetic particles remaining in the suspension can be separated completely. Moreover, when all of the liquid is shifted to the first container, the process can be repeated according to the eighteenth aspect.
According to the sixteenth to the twenty-second aspects, at the time of repeating a simple operation such as passing a large volume of suspension through the liquid passage, or sucking the suspension from the liquid suction passage and discharging the suspension from the liquid discharge passage with respect to all of the suspension, since the magnetic particles in the suspension are automatically separated, all of the magnetic particles in the suspension can be efficiently and easily separated completely with a simple construction.
According to the present invention, concentration can be performed to an optional proportion, by re-suspending the separated magnetic particles in an optional volume of liquid. Therefore, as pre-stage processing of a culture process of bacteria or the like, or of an amplification process of DNA or the like, the density of a large volume of suspension containing a target substance suspended therein in a dilute amount can be increased efficiently or within a short period of time.
Since contact between the suspension containing the target substance such as bacteria suspended therein and the mechanical parts can be avoided or made minimal, the present invention is suitable for handling microorganisms such as bacteria and biopolymers such as DNA substances. Moreover, in the case where the target substance is indirectly captured by the magnetic particles via a carrier, it is not necessary to perform processing such as coating with respect to the magnetic particles, thereby enabling low-cost processing.